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A Metabolomics Investigation of the Physiological Component of the Innate Immune Response in RAW264.7 Macrophages Treated with Nitric Oxide Inhibitors
Author(s) -
May Amanda,
Wepy James,
Marnett Lawrence
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.830.2
Subject(s) - metabolome , metabolomics , nitric oxide , chemistry , arginine , citrulline , biochemistry , glutamine , metabolism , glutathione , metabolite , metabolic pathway , amino acid , enzyme , chromatography , organic chemistry
Ultra performance liquid chromatography‐high resolution mass spectrometry (UPLC‐HRMS) based metabolomics is capable of providing a global snapshot of alterations in the intracellular small molecule content (the metabolome) that can be used to probe the temporal changes in metabolism in response to perturbations. Using untargeted UPLC‐HRMS metabolomics ( Anal. Chem. , 2010 , 82 (8), pp 3212–3221), the metabolome of RAW264.7 macrophages treated with Kdo‐Lipid2 (KLA), a chemically defined lipopolysaccharide, was measured and compared to a control metabolome from untreated cells. Initial biochemical studies were used to supplement the metabolomics data and have shown that treatment of RAW264.7 macrophages with KLA induces nitric oxide synthase (iNOS), which leads to the production of nitric oxide (NO) through the conversion of arginine to citrulline. While arginine concentrations remained unchanged in the two populations, other amino acids involved in nitrogen metabolism (glutamine and glutamate) and oxidative stress (glutathione), show decreases in pool size when cultures are treated with KLA after 8 hours. Metabolites involved in arginine and proline metabolism, the metabolic pathway encompassing NO synthesis, also show a decrease in fold change at 8 h. These are proline, sarcosine, L‐argininosuccinate, and aspartate. Macrophages were also co‐incubated with KLA and either a broad NOS inhibitor, L‐NAME ( Methods Enzymol ., 1996 , 268 , pp 375–392), or selective iNOS inhibitor 1400W ( J Biol Chem ., 1997 , 272 , pp 4959–4963), and the metabolomes and NO production from these cells were recorded. Global alterations in metabolism were noted, and the NO concentrations were diminished at 24 hours, as NO was not detected prior to then, when incubated with either inhibitor. The iNOS activity was also indirectly monitored by measuring citrulline via liquid chromatography‐tandem mass spectrometry. This concentration of this metabolite increased between the KLA treated and untreated populations, while the addition of each inhibitor subsequently diminished the increase at 24 hours. Herein, the temporal metabolic response of RAW264.7 macrophages either treated with KLA or not over 24 hours is reported to reveal differences in metabolism for cells that are experiencing an induction of the inflammatory response via KLA. Support or Funding Information Authors would like to acknowledge funding of this research through the NIEHS (2T32ES007028–40).